The present invention relates to methods for isolating, identifying, quantifying, and propagating chicken infectious anemia virus, in particular, for vaccine production.
Chicken infectious anemia virus (CIAV), also known as chicken anemia virus (CAV) or chicken anemia agent (CAA), belongs to the group of Circoviridae. CIAV was first isolated in Japan in 1979 during an investigation of a Marek""s disease vaccination break (Yuasa et al., Avian Dis. 23:366-385 (1979)). Since that time, CIAV has been detected in commercial poultry in all major poultry producing countries (von Bxc3xclow et al., in Diseases of Poultry, 10th edition, Iowa State University Press, pp. 739-756 (1997)).
CIAV can cause clinical disease, characterized by anemia, hemorrhages and immunosuppression, in young susceptible chickens. Atrophy of the thymus and of the bone marrow are characteristic and consistent lesions of CIAV-infected chickens. Lymphocyte depletion in the thymus, and occasionally in the bursa of Fabricius, results in immunosuppression and increased susceptibility to secondary viral, bacterial, or fungal infections which then complicate the course of the disease. Infection of young chicks (less than 3 weeks) causes anemia, immunosuppression, morbidity and sometimes mortality if the chicks are free of maternal antibodies against CIAV. In chickens with maternal antibodies virus replication is suppressed until the antibodies disappear at approximately 3 weeks of age. After 3 weeks of age, infection is mostly subclinical but may cause changes in production of cytokines affecting the development of optimal immune responses to natural infections and vaccinations. The immunosuppression may cause aggravated disease after infection with one or more of Marek""s disease virus (MDV), infectious bursal disease virus, reticuloendotheliosis virus, adenovirus, or reovirus. It has been reported that pathogenicity of MDV is enhanced by CIAV (deBoer et al., In Proceedings of the 38th Western Poultry Diseases Conference, p. 28, Tempe, Ariz. (1989)). Further, it has been reported that CIAV aggravates the signs of infectious bursal disease (Rosenberger et al., Avian Dis. 33:707-713 (1989)). Additionally, subclinical CIAV infection in older chickens is correlated with decreased performance in broiler flocks (McNulty et al., Avian Dis. 35:263-268 (1991)). CIAV is highly resistant to environmental inactivation and some common disinfectants, characteristics that may potentiate disease transmission. The economic impact of CIAV infection on the poultry industry is reflected by mortality of 10% to 30% in disease outbreaks, a possible role in vaccine failures, and lower performance of infected flocks due to subclinical infection.
CIAV is a small, non-enveloped icosahedral virus of 25 nm diameter, and contains a genome consisting of 2.3 kb circular, single-stranded DNA. Two polypeptides have been detected in purified virus preparations; a major polypeptide of about 50 kilodaltons (kDa) termed VP1, and a 24 kDa polypeptide termed VP2. These two polypeptides together form a major epitope for the production of virus-neutralizing antibodies. Genomic DNA sequences of several different isolates of CIAV have been reported. Isolate Cux-1 was sequenced by Noteborn et al. (Noteborn et al., J. Virol. 65:3131-3139 (1991)) revealing 3 open reading frames (ORFs) that potentially encode polypeptides of 51.6 kDa, 24.0 kDa, and 13.6 kDa. As positioned in the genome, the three ORFs either partially or completely overlap one another. There was only one promoter-enhancer region upstream of the ORFs, and a single polyadenylation signal downstream of the ORFs. A single unspliced mRNA of 2100 bases is transcribed from the Cux-1 genome (Noteborn et al., Gene 118:267-271 (1992)). Another group also sequenced the Cux-1 strain; however, differences were noted between their sequence data and those from Noteborn et al. (Mechan et al., Arch. Virol. 124:301-319 (1992)). The nucleotide sequence of strain 26P4, isolated in the U.S.A., also showed a number of nucleotide differences when compared with sequences of Cux-1 (Claessens et al., J. Gen. Virol. 72:2003-2006 (1991)). Despite the differences in nucleotide sequences found in various isolates from around the world, only minor differences in amino acid sequences have been noted. For these reasons, it has been assumed that CIAV is a highly conserved virus.
Presently, the process by which CIAV causes chicken infectious anemia is poorly understood. When strain CIA-1 is introduced into susceptible 1-day old chicks, CIA-1 produces signs and lesions characteristic of chicken infectious anemia including low hematocrit values, depletion of erythrocytes and lymphoid cells in the bone marrow, depletion of lymphoid cells of the medulla and the cortex of the thymus (herein referred as T-cells), and inflammatory changes in the liver, heart and kidney (Lucio et al., Avian Dis. 34:146-153 (1990)). One or more of the polypeptides encoded by the CIAV ORFs may play a role in the pathogenesis of chicken infectious anemia by facilitating invasion into susceptible cells, and/or initiating T-cell apoptosis.
Exposing hens to CIAV may induce maternal antibody in chickens which may help protect against CIAV infections in their progeny. However, such vaccination with any of the CIAV strains has inherent problems including the potential of vertical (through the egg) transmission, and contamination of the environment. It is therefore desirable to develop a vaccine having as the immunogen a purified polypeptide(s) associated with CIAV.
When CIAV was first isolated in 1979, (Yuasa et al., xe2x80x9cIsolation and Some Characteristics of an Agent Inducing Anemia in Chicks,xe2x80x9d Avian Dis. 23:366-385 (1979)), the only method of propagation was by chick inoculation. The Gifu strain of CIAV was subsequently found by Yuasa et al. (Yuasa, xe2x80x9cPropagation and Infectivity Titration of the Gifu-1 Strain of Chicken Anemia Agent in a Cell Line (MDCC-MSB1) Derived From Marek""s Disease Lymphoma,xe2x80x9d Nat. Inst. Anim. Health Q. 23:13-20 (1983)) to replicate in two lymphoblastoid cell lines, Marek""s disease cell culture (MDCC)-MSB1 (MSB1) (Akiyama et al., xe2x80x9cTwo Cell Lines From Lymphomas of Marek""s Disease,xe2x80x9d Biken J. 17:105-116 (1974)), and MDCC-JP2 (Yamaguchi et al., xe2x80x9cEstablishment of Marek""s Disease Lymphoblastoid Cell Lines from Chickens with BABK of B Blood Groups,xe2x80x9d Biken J. 22:35-40 (1979)), and the lymphoblastoid avian leukosis virus-transformed cell line, LSCC-1104X5 (Hihara et al., xe2x80x9cEstablishment of Tumor Cell Lines Cultured From Chickens With Avian Lymphoid Leukosis,xe2x80x9d Nat. Inst. Anim. Health Q. 14:163-173 (1974)). Two other Marek""s disease cell lines, MDCC-RP1 (Nazerian et al., xe2x80x9cA Nonproducer T Lymphoblastoid Cell Line From Marek""s Disease Transplantable Tumor (JMV),xe2x80x9d Avian Dis. 21:69-76 (1977)) and MDCC-BP1 (Yuasa, xe2x80x9cPropagation and Infectivity Titration of the Gifu-1 Strain of Chicken Anemia Agent in a Cell Line (MDCC-MSB1) Derived From Marek""s Disease Lymphoma,xe2x80x9d Nat. Inst. Anim. Health Q. 23:13-20 (1983)), and one lymphoid leukosis line, LSCC-TLT-1 (current terminology: LSCC-CU 10) (Calnek et al., xe2x80x9cEstablishment of Marek""s Disease Lymphoblastoid Cell Lines From Transplantable Versus Primary Lymphomas,xe2x80x9d Int. J. Cancer 21:100-197 (1978)) apparently failed to support the growth of the virus. More recent reports (Chandratilleke et al., xe2x80x9cCharacterization of Proteins of Chicken Infectious Anemia Virus with Monoclonal Antibodies,xe2x80x9d Avian Dis. 35:854-862 (1991) and Renshaw et al., xe2x80x9cA Hypervariable Region in VP1 of Chicken Infectious Anemia Virus Mediates Rate of Spread and Cell Tropism in Tissue Culture,xe2x80x9d J. Virology 70:8872-8878 (1996)) suggest that other MD cell lines such as MDCC-CU22 (Calnek et al., xe2x80x9cSpontaneous and Induced Herpesvirus Genome Expression in Marek""s Disease Tumor Cell Lines,xe2x80x9d Infect. Immun. 34:483-491 (1981)) and a reticuloendotheliosis virus-transformed T-cell line, RECC-CU205 (Schat et al, xe2x80x9cStable Transfection of Reticuloendotheliosis Virus-Transformed Lymphoblastoid Cell Lines,xe2x80x9d Avian Dis. 36:432-439 (1992)), also are susceptible to one or more strains of CIAV. The virus also can be propagated in chicken embryos (von Bxc3xclow et al., xe2x80x9cChicken Vermehrung des Erregers der Avixc3xa4ren Infektiosen Anxc3xa4mie (CAA) in Embryonierten Hxc3xchnereiem,xe2x80x9d J. Vet. Med. B 33:664-669 (1986)).
MSB1 cells, characterized as mature helper T lymphocytes (CD3+, CD4+, CD8xe2x88x92, TCR2+) (Adair et al., xe2x80x9cCharacterization of Surface Markers Present on Cells Infected by Chicken Anemia Virus in Experimentally Infected Chickens,xe2x80x9d Avian Dis. 37:943-950 (1993)), are the most commonly reported substrate used for in vitro isolation, propagation, and titration of CIAV (von Bxc3xclow et al., xe2x80x9cChicken Infectious Anemia,xe2x80x9d Diseases of Poultry, 10th ed., Iowa State University Press, pp. 739-756 (1997) and McNulty, xe2x80x9cChicken Anaemia Agent: a Review,xe2x80x9d Avian Pathol. 20:187-203 (1991)). Criteria of infection of MSB1 cultures include cytopathic effects and detection of viral antigen(s) by immunofluorescence (IF) tests or other methods. Although these cells appear to be the preferred substrate for in vitro infection with many strains of CIAV, some virus strains have been reported to not infect certain sublines of MSB1, or to do so only poorly. For instance, Cux-1 (von Bxc3xclow et al., xe2x80x9cFrxc3xchsterblichkeitssyndrom bei Kxc3xcken nach Doppelinfektion mit dem Virus der Marekshen Krankheit (MDV) und einem Anxc3xa4mi-Erreger (CAA),xe2x80x9d Veterinaermed Reihe B 30:742-750 (1983)), CIA-1 (Lucio et al., xe2x80x9cIdentification of the Chicken Anemia Agent, Reproduction of the Disease, and Serological Survey in the United States,xe2x80x9d Avian Dis. 34:146-153 (1990)), and L-028 (Renshaw et al., xe2x80x9cA Hypervariable Region in VP1 of Chicken Infectious Anemia Virus Mediates Rate of Spread and Cell Tropism in Tissue Culture,xe2x80x9d J. Virology 70:8872-8878 (1996)) all were found to replicate in one subline of MSB1, MSB1(S), but only Cux-1 replicated in a second subline, MSB1(L) and then to a lesser degree than in MSB12 (S). Furthermore, strain CIA-1 grew more slowly than Cux-1 in MSB12(S) cells. In a preliminary report by Lucio et al. in 1992 (Lucio-Martinez et al, xe2x80x9cComparative Susceptibility of Avian Cell Lines to Chicken Infectious Anemia Virus (abstract),xe2x80x9d Proc. 129th Ann. Meet. Amer. Vet. Med. Assoc. Boston, Mass. (1992)), there appeared to be substantial differences in CIAV-susceptibility among cell lines with some lines appearing to be more susceptible than MSB1(L) to the Cux-1 strain of CIAV.
The present invention is directed to overcoming the deficiencies in the prior art in isolating, identifying, quantifying, and propagating chicken infectious anemia virus.
The present invention relates to a method of propagating chicken infectious anemia virus. This method involves providing a Marek""s disease chicken cell linexe2x80x94CU147 culture and inoculating the culture with a chicken infectious anemia virus under conditions effective to propagate the virus in the culture.
The present invention also relates to a method of isolating chicken infectious anemia virus from a sample. This method involves providing a biological sample infected with a chicken infectious anemia virus, providing a Marek""s disease chicken cell linexe2x80x94CU147 culture, incubating the culture with the biological sample under conditions effective to allow the virus to infect the culture, and isolating the virus from the culture.
Another aspect of the present invention is a method for identifying chicken infectious anemia virus in a sample. This method involves providing a biological sample potentially containing a chicken infectious anemia virus, providing a Marek""s disease chicken cell linexe2x80x94CU147 culture, incubating the culture with the biological sample under conditions effective to allow any of the virus present in the biological sample to infect the culture, and identifying the presence of any of the virus in the culture.
Yet another aspect of the present invention is a method for quantifying chicken infectious anemia virus in a sample. This method involves providing a biological sample containing a quantity of chicken infectious anemia virus, providing a Marek""s disease chicken cell linexe2x80x94CU147 culture, incubating the culture with the biological sample under conditions effective to allow the virus to infect the culture, and titrating the quantity of virus in the culture.
The present invention also relates to a high titer vaccine formulation for chicken infectious anemia virus which includes an immunologically effective amount of chicken infectious anemia virus propagated in a Marek""s disease chicken cell linexe2x80x94CU147 culture.
Another aspect of the present invention is a method of immunizing poultry against chicken infectious anemia virus which includes administering a vaccine prepared from chicken infectious anemia virus propagated in a Marek""s disease chicken cell linexe2x80x94CU147 culture in an amount effective to induce an immune response to the virus.
The methods of the present invention can be used to replicate virus to higher titers than with prior art methods. In addition, the use of the methods of the present invention for the production of a vaccine results in improved yields of virus and, therefore, improved vaccine production. Moreover, the methods of the present invention can be used to produce high yields of virus and virus antigen to be used in diagnostic assays.
The present invention relates to a method of propagating chicken infectious anemia virus (CIAV). This method involves providing a Marek""s disease chicken cell linexe2x80x94CU147 (MDCC-CU147) culture (ATCC Accession No. PTA-1476) and inoculating the culture with a chicken infectious anemia virus under conditions effective to propagate the virus in the culture.
Suitable virus strains of CIAV include, but are not limited to, CIA-1 strain (GenBank Accession No. L14767, which is hereby incorporated by reference), Cux-1strain (GenBank Accession No. M55918, which is hereby incorporated by reference), Gifu strain (Yuasa, xe2x80x9cPropagation and Infectivity Titration of the Gifu-1 Strain of Chicken Anemia Agent in a Cell Line (MDCC-MSB1) Derived From Marek""s Disease Lymphoma,xe2x80x9d Nat. Inst. Anim. Health Q. 23:13-20 (1983), which is hereby incorporated by reference), TK-5803 strain (Goryo et al., xe2x80x9cSerial Propagation and Purification of Chicken Anaemia Agent in MDCC-MSB1 Cell Line,xe2x80x9d Avian Pathology 16:149-163 (1987), which is hereby incorporated by reference), CAA82-2 strain (Otaki et al., xe2x80x9cIsolation of Chicken Anaemia Agent and Marek""s Disease Virus from Chickens Vaccinated with Turkey Herpesvirus and Lesions Induced in Chicks by Inoculating Both Agents,xe2x80x9d Avian Pathology 16:291-306 (1987), which is hereby incorporated by reference), L-028 strain (ORF1: GenBank Accession No. U69549, which is hereby incorporated by reference), Conn strain (ConnB: ORF1: GenBank Accession No. U69548, which is hereby incorporated by reference), GA strain (Goodwin et al., xe2x80x9cIsolation and Identification of a Parvovirus-Like Virus (The So-Called Chick Anemia Agent (CAA)) that Causes Infectious Anemia in Chicks,xe2x80x9d Proc. 38th Western Poultry Disease Conference, Tempe, Ariz., pp. 21-23 (1989), which is hereby incorporated by reference), 26P4 strain (GenBank Accession No. I-1141, which is hereby incorporated by reference), SR43 strain (Zhou et al., xe2x80x9cIsolation and Identification of Chicken Infectious Anemia Virus in China,xe2x80x9d Avian Diseases 41:361-364 (1997), which is hereby incorporated by reference), and CL-1 strain (Lamichhane et al., xe2x80x9cPathogenicity of CL-1 Chicken Anemia Agent,xe2x80x9d Avian Diseases 35:515-522 (1991), which is hereby incorporated by reference).
Cell lines such as the MDCC-CU147 cell line can be provided by numerous techniques known to those of ordinary skill in the art. In particular, the MDCC-CU147 cell line can be derived from Marek""s disease lymphomas induced in chickens. Virus strains which can be used to induce tumors include: the low-oncogenicity strain CU-2 (Smith et al., xe2x80x9cEffect of Virus Pathogenicity on Antibody Production in Marek""s Disease,xe2x80x9d Avian Dis. 17:727-736 (1973), which is hereby incorporated by reference); moderate-oncogenicity strains BC-1 (Murthy et al., xe2x80x9cPathogenesis of Marek""s Disease: Early Appearance of Marek""s Disease Tumor-Associated Surface Antigen in Infected Chickens,xe2x80x9d J. Natl. Cancer Inst. 61:849-854 (1978), which is hereby incorporated by reference), ConnB (Jakowski et al., xe2x80x9cHematopoietic Destruction in Marek""s Disease Viruses in Chickens,xe2x80x9d Avian Dis. 14:374-385 (1970), which is hereby incorporated by reference), and JM-10 (Calnek, xe2x80x9cInfluence of Age at Exposure on the Pathogenesis of Marek""s Disease,xe2x80x9d J. Natl. Cancer Inst. 51:929-939 (1973), which is hereby incorporated by reference); high-oncogenicity strain GA-5 (Calnek, xe2x80x9cInfluence of Age at Exposure on the Pathogenesis of Marek""s Disease,xe2x80x9d J. Natl. Cancer Inst. 51:929-939 (1973), which is hereby incorporated by reference); and the very high-oncogenicity strain RB-1B (Schat et al., xe2x80x9cInfluence of Oncogenicity of Marek""s Disease Virus on Evaluation of Genetic Resistance,xe2x80x9d Poult. Sci. 60:2559-2566 (1981), which is hereby incorporated by reference).
Further, cell lines such as the MDCC-CU147 cell line can be established from early local lesions induced by Marek""s disease virus and alloantigens as described in Calnek et al., xe2x80x9cPathogenesis of Marek""s Disease Virus-Induced Local Lesions. 2. Influence of Virus Strain and Host Genotype,xe2x80x9d In: Advances in Marek""s Disease Research, Kato et al., Eds., Gapanses Association on Marek""s Disease, Osaka, Japan, pp. 324-330 (1988) and Calnek et al., xe2x80x9cPathogenesis of Marek""s Disease Virus-Induced Local Lesions. 1. Lesion Characterization and Cell Line Establishment,xe2x80x9d Avian Dis. 33:291-302 (1989), which are hereby incorporated by reference.
The preparation and maintenance of cultures of the MDCC-CU147 cell line may be effected by techniques which are well known in the art. For example, cultures may be seeded at 250,000 cells/ml in plastic flasks or in 24-well plastic plates in an appropriate medium, such as LM Hahn medium or Leibovitz""s L-1 5-McCoy""s 5A medium (Calnek et al., xe2x80x9cSpontaneous and Induced Herpesvirus Genome Expression in Marek""s Disease Tumor Cell Lines,xe2x80x9d Infect. Immun. 34:483-491 (1981), which is hereby incorporated by reference), and then incubated, e.g., in a 5% CO2 atmosphere at approximately 40-41xc2x0 C.
In a preferred embodiment, inoculation is at a level from about 20 xcexcL undiluted virus/ml culture to about 100 xcexcL undiluted virus/ml culture.
In the method of the present invention, MDCC-CU147 can be used to replicate the CIAV to higher titers than in sublines of MSB-1 (see Tables 2, 3, and 4 in the Examples, below). These results could not be expected based on the phenotype of MDCC-CU147 because other cell lines with a similar phenotype (Tables 1 and 3) are much less susceptible to infection with and the replication of CIAV. In addition, the use of MDCC-CU147 instead of other cell lines (e.g., MSB-1) for the production of a CIAV vaccine results in improved yields of virus providing a competitive advantage to any company using MDCC-CU147 for vaccine production or other purposes. In particular, the use of MDCC-CU147 allows production of high yields of virus and virus antigen to be used in diagnostic assays.
The present invention also relates to a method of isolating chicken infectious anemia virus from a sample. This method involves providing a biological sample infected with a chicken infectious anemia virus, providing a Marek""s disease chicken cell linexe2x80x94CU147 culture, incubating the culture with the biological sample under conditions effective to allow the virus to infect the culture, and isolating the virus from the culture.
Suitable biological samples include blood, mucosal scrapings, semen, tissue biopsy, embryonal tissues, secretions and excretions, and swabs of bodily fluids.
The virus may be isolated from the infected cells of the culture using methods known to those of ordinary skill in the art. In particular, virus can be isolated from infected cells by co-cultivation of infected cells with MDCC-CU147 culture cells, or from extracts of infected cells obtained by any of the typical methods for virus extraction, such as sonication, centrifugation, and freeze-thaw, or from secretions, excretions, or swabs of other bodily fluids.
Another aspect of the present invention is a method for identifying chicken infectious anemia virus in a sample. This method involves providing a biological sample potentially containing a chicken infectious anemia virus, providing a Marek""s disease chicken cell linexe2x80x94CU147 culture, incubating the culture with the biological sample under conditions effective to allow any of the virus present in the biological sample to infect the culture, and identifying the presence of any of the virus in the culture.
Suitable methods for identifying the presence of the virus in the culture, i.e., demonstrating the presence of viral proteins in the culture, include immunofluorescence tests, which may use a monoclonal antibody against one of the viral proteins or polyclonal antibodies (von Bxc3xclow et al., in Diseases of Poultry, 10th edition, Iowa State University Press, pp. 739-756 (1997), which is hereby incorporated by reference), polymerase chain reaction (PCR) or nested PCR (Soinxc3xa9 et al., Avian Diseases 37:467-476 (1993), which is hereby incorporated by reference), ELISA (von Bxc3xclow et al., in Diseases of Poultry, 10th edition, Iowa State University Press, pp. 739-756 (1997), which is hereby incorporated by reference), virus neutralization, or any of the common histochemical methods of identifying specific viral proteins.
The method of identifying chicken infectious anemia virus in a sample of the present invention is particularly applicable to the development of diagnostic tests. In particular, once the presence of the virus in the culture is identified, the viral proteins may be extracted from the culture and used as a substrate for a diagnostic test, e.g., ELISA, immunofluorescence techniques, and PCR techniques.
Yet another aspect of the present invention is a method for quantifying chicken infectious anemia virus in a sample. This method involves providing a biological sample containing a quantity of chicken infectious anemia virus, providing a Marek""s disease chicken cell linexe2x80x94CU147 culture, incubating the culture with the biological sample under conditions effective to replicate the virus in the culture, and titrating the quantity of the virus in the culture.
Titrating the quantity of the virus in the culture may be effected by techniques known in the art, as described in Villegas et al., xe2x80x9cTitration of Biological Suspensions,xe2x80x9d In: A Laboratory Manual for the Isolation and Identification of Avian Pathogens, 3rd Ed., Purchase et al., Eds., Kendall/Hunt Publishing Co., Dubuque, Iowa, pp. 186-190 (1989), which is hereby incorporated by reference.
The present invention also relates to a high titer vaccine formulation for chicken infectious anemia virus which includes an immunologically effective amount of chicken infectious anemia virus propagated in a Marek""s disease chicken cell linexe2x80x94CU147 culture.
CIAV can be cultured in the MDCC-CU147 culture to a titer of at least 5xc3x97107 tissue culture infective dosesxe2x80x94fifty percent (TCID50).
One embodiment of the present invention is a live vaccine. Live attenuated vaccines may be produced by methods known in the art. For example, live attenuated vaccines may be produced by passaging and propagating the CIAV in an appropriate cell culture, e.g., the MDCC-CU147 culture, followed by subsequent propagation and passaging in embryonated eggs (see U.S. Pat. No. 5,728,569 to Schrier et al., which is hereby incorporated by reference). The vaccines of the present invention containing a live attenuated CIAV strain can be prepared and marketed in the form of a suspension or as a lyophilized product in a manner known per se.
Another embodiment of the present invention is an inactivated vaccine which includes one or more isolates of inactivated CIAV propagated in an MDCC-CU147 culture.
Inactivation of CIAV (to eliminate reproduction of the virus) for use in the vaccine of the present invention can be achieved, in general, by chemical or physical means (see U.S. Pat. No. 5,728,569 to Schrier et al., which is hereby incorporated by reference). Chemical inactivation can be effected by treating the virus with, for example, enzymes, formaldehyde, beta-propiolactone, ethylene-imine, or a derivative thereof. If necessary, the inactivating compound can be neutralized after inactivation is complete. For example, material inactivated with formaldehyde can be neutralized with thiosulfate. Physical inactivation can be effected by subjecting the virus to energy-rich radiation, e.g., UV light, X-radiation, or gamma-radiation. If desired, the pH can be brought back to a value of about 7 after treatment.
The vaccines of the present invention are administered in a dose sufficient to induce an immune response to the CIAV (see U.S. Pat. No. 5,728,569 to Schrier et al., which is hereby incorporated by reference).
The vaccines of the present invention can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. They can be administered alone or with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, and can be in solid or liquid form such as, powders, solutions, suspensions, or emulsions.
The CIAV propagated in a Marek""s disease chicken cell linexe2x80x94CU147 culture of the present invention may be administered in injectable dosages by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carriers, including adjuvants, excipients, or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
For use as aerosols, the CIAV propagated in a Marek""s disease chicken cell linexe2x80x94CU147 culture of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
As described above, a stabilizer may be added to the vaccine composition. Suitable stabilizers include SPGA (Bavarnik et al., J. Bacteriology, 59:509-522 (1950), which is hereby incorporated by reference), carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, or glucose), proteins (such as albumin or casein), or degradation products thereof, and buffers (such as alkali metal phosphates).
In addition, suitable adjuvants can also be added to the vaccine formulation. Suitable compounds with adjuvant activity include vitamin-E acetate oil-in-water emulsion, aluminum hydroxide, phosphate, or oxide, mineral oil (e.g., BAYOL(copyright) and MARCOL(copyright)), and saponins.
Emulsifiers, such as TWEEN(copyright) and SPAN(copyright), may also be added to the vaccine formulation.
Vaccines according to the present invention may contain combinations of the CIAV propagated in an MDCC-CU147 culture and one or more unrelated avian viruses. Suitable unrelated avian viruses include Newcastle Disease virus (xe2x80x9cNDVxe2x80x9d), Infectious Bronchitis virus (xe2x80x9cIBVxe2x80x9d) (ATCC Accession Nos. VR-21, VR-22, VR-817, and VR-841), Infectious Bursal Disease virus (IBVD) (ATCC Accession Nos. VR-478, VR-2041, and VR-2161), Marek""s Disease virus (xe2x80x9cMDVxe2x80x9d) (ATCC Accession Nos. VR-585, VR-624, VR-987, VR-2001, VR-2002, VR-2103, VR-2175, VR-2176, and VR-2260), Herpes virus of Turkeys (xe2x80x9cHVTxe2x80x9d) (ATCC Accession No. VR-584B), Infectious Laryngotracheitis virus (ATCC Accession No. VR-783) or other avian herpes, Reo virus, Egg Drop Syndrome virus, Avian Encephalomyelitis virus (ATCC Accession Nos. VR-713 and VR-2058), Reticuloendotheliosis virus (ATCC Accession Nos. VR-770, VR-994, 45011, 45012, 45013, and 45158), Leukosis virus (ATCC Accession Nos. VR-247, VR-335, VR-658, and VR-773), Fowlpox virus (ATCC Accession Nos. VR-229, VR-249, VR-250, and VR-251), Turkey Rhinotracheitis virus (xe2x80x9cTRTVxe2x80x9d), Adenovirus, or Avian Influenza virus (ATCC Accession No. VR-40).
Another aspect of the present invention is a method of immunizing poultry against chicken infectious anemia virus which includes administering a vaccine prepared from chicken infectious anemia virus propagated in a Marek""s disease chicken cell linexe2x80x94CU147 culture in an amount effective to induce an immune response to the virus.
This method includes the administration of live or inactivated vaccines.